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Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes.

Purpose To characterize corneal subbasal nerve plexus features of normal and simian immunodeficiency virus (SIV)-infected macaques by combining in vivo corneal confocal microscopy (IVCM) with automated assessments using deep learning-based methods customized for macaques. Methods IVCM images were collected from both male and female age-matched rhesus and pigtailed macaques housed at the Johns Hopkins University breeding colony using the Heidelberg HRTIII with Rostock Corneal Module. We also obtained repeat IVCM images of 12 SIV-infected animals including preinfection and 10-day post-SIV infection time points. All IVCM images were analyzed using a deep convolutional neural network architecture developed specifically for macaque studies. Results Deep learning-based segmentation of subbasal nerves in IVCM images from macaques demonstrated that corneal nerve fiber length and fractal dimension measurements did not differ between species, but pigtailed macaques had significantly higher baseline corneal nerve fiber tortuosity than rhesus macaques (P = 0.005). Neither sex nor age of macaques was associated with differences in any of the assessed corneal subbasal nerve parameters. In the SIV/macaque model of human immunodeficiency virus, acute SIV infection induced significant decreases in both corneal nerve fiber length and fractal dimension (P = 0.01 and P = 0.008, respectively). Conclusions The combination of IVCM and robust objective deep learning analysis is a powerful tool to track sensory nerve damage, enabling early detection of neuropathy. Adapting deep learning analyses to clinical corneal nerve assessments will improve monitoring of small sensory nerve fiber damage in numerous clinical settings including human immunodeficiency virus.

This study examined the relationship between maternal food source and preparation during pregnancy and the duration of breastfeeding among 751 mother–child dyads in the United States. The data collected from the Environmental influences on Child Health Outcomes (ECHO) Program included twelve cohorts of mothers (age ≥ 18) who delivered infant(s). Three categories of maternal food source and preparation including, High, Moderate, or Low Food Source Quality were derived from the mother report. The mean duration of breastfeeding differed strongly across the three categories. The High Food Source Quality group breastfed an average of 41 weeks, while shorter durations were observed for the Moderate (26 weeks) and Low (16 weeks) Food Source Quality groups. Cox proportional hazards models were used to estimate the relative hazard of time to breastfeeding cessation for each participant characteristic. The full model adjusted for clustering/cohort effect for all participant characteristics, while the final model adjusted for the subset of characteristics identified from variable reduction modeling. The hazard of breastfeeding cessation for those in the High Food Source Quality group was 24% less than the Moderate group (RH = 0.76; 95% CI, 0.63–0.92). Pregnant women in the High Food Source Quality group breastfed longer than the Moderate and Low groups. We encourage more detailed studies in the future to examine this relationship longitudinally.

Background. Disrupted sleep is common after traumatic brain injury (TBI) particularly in the inpatient rehabilitation setting where it may affect participation in therapy and outcomes. Treatment of sleep disruption in this setting is varied and largely unexamined. Objective. To study the feasibility of instituting a sleep hygiene intervention on a rehabilitation unit. Methods. Twenty-two individuals admitted to a brain injury unit were enrolled and allocated, using minimization, to either a sleep hygiene protocol (SHP) or standard of care (SOC). All participants wore actigraphs, underwent serial cognitive testing, and had light monitors placed in their hospital rooms for 4 weeks. Additionally, participants in the SHP received 30 minutes of blue-light therapy each morning, had restricted caffeine intake after noon, and were limited to 30-minute naps during the day. SHP participants had their lights out time set according to preinjury sleep time preference. Both groups were treated with the same restricted formulary of centrally acting medications. Results. Of 258 patients screened, 27 met all study inclusion criteria of whom 22 were enrolled. Nine participants in each group who had at least 21 days of treatment were retained for analysis. The protocol was rated favorably by participants, families, and staff. Actigraph sleep metrics improved in both groups during the 4-week intervention; however, only in the SHP was the change significant. Conclusions. Sleep hygiene is a feasible, nonpharmacologic intervention to treat disrupted sleep in a TBI inpatient rehabilitation setting. A larger study is warranted to examine treatment efficacy. ClinicalTrials.gov Identifier: NCT02838082.

To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). Results were compared with standard EV characterization techniques such as transmission electron microscopy (TEM) and Western blot (WB). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized per MISEV2018 guidelines. Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode and NFCM were able to detect at least two markers on the same particle. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Background: No study has attempted to associate the levels of preinjury serum biomarkers of collagen turnover with the subsequent risk of anterior cruciate ligament (ACL) injury. Hypothesis: Preinjury serum biomarkers of collagen turnover would be associated with the subsequent risk of ACL injury. Study Design: Case-control study; Level of evidence, 3. Methods: We conducted a case-control study with 45 ACL-injured cases and 45 controls matched for sex, age, height, and weight. In addition to the matching criteria, controls had no history of major joint injury. Baseline preinjury serum samples were obtained from the Department of Defense Serum Repository for all subjects. Samples were assessed for 2 serum biomarkers of collagen synthesis (CPII and CS846) and 2 markers of collagen degradation (C1,2C and C2C) through commercially available enzyme-linked immunosorbent assay (ELISA) kits. All ELISAs were performed in triplicate. Conditional logistic regression models were used to analyze the data. Results: Univariate results suggested that both biomarkers for collagen degradation (C1,2C and C2C) were significantly associated with the subsequent likelihood of ACL injury. Serum C2C and C1,2C concentration at baseline were associated with odds ratios (ORs) of 2.05 (95% CI, 1.30-3.23; P = .001) and 3.02 (95% CI, 1.60-5.71; P = .002), respectively. Baseline serum CPII concentrations were also associated with subsequent ACL injury. Serum CPII concentration at baseline was associated with an OR of 4.41 (95% CI, 1.87-10.38; P = .001). Baseline serum CS846 levels approached significance (OR = 0.77; 95% CI, 0.57-1.03; P = .080). Multivariable models suggested that preinjury CPII and C2C concentrations at baseline are important indicators of subsequent ACL injury risk. Conclusion: Preinjury differences in serum biomarker levels of collagen turnover suggest that collagen metabolism in individuals who go on to tear an ACL may be different when compared with a matched control group with no history of major joint injury. These differences may be reflective of different preinjury biochemical and/or biomechanical risk profiles or genetic factors that subsequently affect both collagen metabolism and ACL injury risk.

Background As awareness of disrupted sleep in patients with traumatic brain injury (TBI) increases so does interest in finding objective measures of sleep. As a result, many clinicians are turning to actigraphs to monitor sleep in patients with altered consciousness. Actigraphs are accelerometers which have been used in sleep research for over four decades. Objective The purpose of the present study was to determine the best method for scoring actigraphs in a TBI population and to describe the benefits and pitfalls of using actigraphs with patients on a brain injury rehabilitation unit. Methods A retrospective chart review of 43 patients compared three different ways of scoring night time rest periods: autoscoring, manual scoring, and set interval scoring for the sleep parameters of sleep efficiency, wakefulness after sleep onset, and total sleep time. Nursing compliance with using the event marker on the device to set rest period was also analyzed. Results The autoscoring method of determining the rest interval showed an inflation of sleep efficiency. For each sleep parameter compared, the strongest correlations were observed between the manual and set interval scoring methods. Compliance using event markers to set rest interval was low (16.7%). Conclusions Set interval scoring is the most efficient method to determine the rest interval in TBI patients. The use of event markers was an unreliable method to determine rest period.

In this study, total coliforms (TC), Escherichia coli, enterovirus (EV), rotavirus (RV), and human mastadenovirus species C and F (HAdV-C and HAdV-F) were evaluated in water samples from Belo Stream. For HAdV-C and F, the infectivity was assessed by integrated cell culture quantitative real-time polymerase chain reaction (ICC-qPCR). Samples were collected monthly (May/2015 to April/2016) at four sites. Viral analyses were performed for both ultracentrifuge-concentrated and unconcentrated samples. For site P4 (used for recreational purposes), QMRA was applied to estimate health risks associated with exposure to E. coli and HAdV-C and F. TC and E. coli were present throughout the collection period. EV and RV were not detected. HAdV-C were present in 8.51% (1.89E + 06 to 2.28E + 07 GC (Genomic Copies)/L) and 21.27% (2.36E + 05 to 1.29E + 07 GC/L) for unconcentrated and concentrated samples, respectively. For HAdV-F were 12.76% (2.77E + 07 to 3.31E + 08 GC/L) and 48.93% (1.10E + 05 to 4.50E + 08 GC/L) for unconcentrated and concentrated samples, respectively. For unconcentrated samples, infectivity for HAdV-C was detected in 37.20% (1st ICC-qPCR) and 25.58% (2nd ICC-qPCR). For HAdV-F, infectivity was detected in 6.97% (1st ICC-qPCR) and 6.97% (2nd ICC-qPCR). For concentrated samples, HAdV-C infectious was observed in 17.02% (1st ICC-qPCR) and in 8.51% (2nd ICC-qPCR). For HAdV-F, were present in 8.51% for both 1st and 2nd ICC-qPCR. Statistical analyzes showed significant difference between the collection sites when analyzed the molecular data of HAdV-F, data of TC and E. coli. Correlation tests showed direct correlation between HAdV-F with E. coli and TC. E. coli concentrations translated to the lowest estimates of infection risks (8.58E-05 to 2.17E-03). HAdV-F concentrations were associated with the highest infection risks at 9.99E-01 and for group C, 1.29E-01 to 9.99E-01. These results show that commonly used bacterial indicators for water quality may not infer health risks associated with viruses in recreational freshwaters.

Staphylococcus aureus nasal carriage is transient in most humans and usually benign, but dissemination of S. aureus to extranasal sites causes the majority of clinical infections, and S. aureus is a major cause of serious infections in the United States. A better understanding of innate nasal decolonization mechanisms is urgently needed, as are relevant models for studying S. aureus clearance. Here, we screened a population of healthy smokers for nasal S. aureus carriage and compared the participants' abilities to clear experimentally applied nasal S. aureus before and after completion of a smoking cessation program. We determined that cigarette smoking increases the mean nasal S. aureus load (2.6 × 10 4 CFU/swab) compared to the load observed in healthy nonsmokers (1.7 × 10 3 CFU/swab) and might increase the rate of S. aureus nasal carriage in otherwise-healthy adults: 22 of 99 smokers carried S. aureus at the screening visit, while only 4 of 30 nonsmokers screened positive during the same time period. Only 6 of 19 experimental inoculation studies in active smokers resulted in S. aureus clearance within the month of follow-up, while in the cessation group, 6 of 9 subjects cleared nasal S. aureus and carriage duration averaged 21 ± 4 days. Smoking cessation associated with enhanced expression of S. aureus -associated interleukin-1β (IL-1β) and granulocyte colony-stimulating factor (G-CSF) in nasal fluids. Participants who failed to clear S. aureus exhibited a higher nasal S. aureus load and elevated nasal interleukin-1 receptor antagonist (IL-1RA) expression at the preexperiment study visits. We conclude that smokers exhibit higher S. aureus loads than nonsmokers and that innate immune pathways, including G-CSF expression and signaling through the IL-1 axis, are important mediators of nasal S. aureus clearance.

Activation of the kynurenine pathway (KP) of tryptophan catabolism likely contributes to HIV-associated neurological disorders. However, KP activation in brain tissue during HIV infection has been understudied, and the effect of combination antiretroviral therapy (cART) on KP induction in the brain is unknown. To examine these questions, tryptophan, kynurenine, 3-hydroxykynurenine, quinolinic acid, and serotonin levels were measured longitudinally during SIV infection in the striatum and CSF from untreated and cART-treated pigtailed macaques. Messenger RNA (mRNA) levels of KP enzymes also were measured in the striatum. In untreated macaques, elevations in KP metabolites coincided with transcriptional induction of upstream enzymes in the KP. Striatal KP induction was also temporally associated-but did not directly correlate-with serotonin losses in the brain. CSF quinolinic acid/tryptophan ratios were found to be the earliest predictor of neurological disease in untreated SIV-infected macaques, outperforming other KP metabolites as well as the putative biomarkers interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Finally, cART did not restore KP metabolites to control levels in the striatum despite the control of the virus, though CSF metabolite levels were normalized in most animals. Overall, these results demonstrate that cerebral KP activation is only partially resolved with cART and that CSF QUIN/TRP ratios are an early, predictive biomarker of CNS disease.

Immune pressure exerted by MHC class I-restricted cytotoxic T cells drives the development of viral escape mutations, thereby regulating HIV disease progression. Nonetheless, the relationship between host immunity and HIV central nervous system (CNS) disease remains poorly understood. The simian immunodeficiency virus (SIV) macaque model recapitulates key features of HIV infection including development of AIDS and CNS disease. To investigate cell-mediated immunity regulating SIV CNS disease progression, we compared the incidence of SIV encephalitis and the influence of MHC class I allele expression on the development of CNS disease in rhesus macaques (Macaca mulatta) versus pigtailed macaques (Macaca nemestrina). After inoculation with the immunosuppressive swarm SIV/DeltaB670 and the neurovirulent molecular clone SIV/17E-Fr, pigtailed macaques progressed more rapidly to AIDS, had higher plasma and cerebrospinal fluid (CSF) viral loads, and were more likely to progress to SIV-associated encephalitis (SIVE) compared to rhesus macaques. In addition, MHC class I alleles were neuroprotective in both species (Mamu-A*001 in rhesus macaques and Mane-A1*084:01:01 in pigtailed macaques); animals expressing these alleles were less likely to develop SIV encephalitis and correspondingly had lower viral replication in the brain. Species-specific differences in susceptibility to SIV disease demonstrated that cell mediated immune responses are critical to SIV CNS disease progression.

Macrophages are targets of HIV-1 infection, and control of viral replication within these cells may be an important component of a T-cell-based vaccine. Although several studies have analyzed the ability of CD8+ T cells to inhibit viral replication in monocyte-derived macrophages, the effect of T cells on HIV-1-infected tissue macrophages is less clear. We demonstrate here that both CD4+ and CD8+ T-cell effectors from HIV controllers are capable of suppressing viral replication in bronchoalveolar lavage-derived alveolar macrophages. These findings have implications for HIV-1 vaccine and eradication strategies.

Chronic microglial activation and associated neuroinflammation are key factors in neurodegenerative diseases including HIV-associated neurocognitive disorders. Colony stimulating factor 1 receptor (CSF1R)-mediated signaling is constitutive in cells of the myeloid lineage, including microglia, promoting cell survival, proliferation, and differentiation. In amyotrophic lateral sclerosis and Alzheimers disease, CSF1R is upregulated. Inhibiting CSF1R signaling in animal models of these diseases improved disease outcomes. In our studies, CNS expression of the CSF1R ligand, colony-stimulating factor 1 (CSF1) was significantly increased in a SIV/macaque model of HIV CNS disease. Using a Nanostring nCounter immune panel, we found CSF1 overexpression was strongly correlated with upregulation of microglial genes involved in antiviral and oxidative stress responses. Using in situ hybridization, we found that CSF1R mRNA was only present in Iba-1 positive microglia. By ELISA and immunostaining with digital image analysis, SIV-infected macaques had significantly higher CSF1R levels in frontal cortex than uninfected macaques (p = 0.018 and p = 0.02, respectively). SIV-infected macaques treated with suppressive ART also had persistently elevated CSF1R similar to untreated SIV-infected macaques. Coordinate upregulation of CSF1 and CSF1R expression implicates this signaling pathway in progressive HIV CNS disease.

Evaluating the effect of variables on diagnostic measures (sensitivity, specificity, positive, and negative predictive values) is often of interest to clinical researchers. Logistic regression (LR) models can be used to predict diagnostic measures of a screening test. A marginal model framework using generalized estimating equation (GEE) with logit/log link can be used to compare the diagnostic measures between two or more screening tests. These individual modeling approaches to each diagnostic measure ignore the dependency among these measures that might affect the association of covariates with each diagnostic measure. The diagnostic measures are computed using joint distribution of screening test result and reference test result which generates a multinomial response data. Thus, multinomial logistic regression (MLR) is a more appropriate approach to modeling these diagnostic measures. In this study, the validity of LR and GEE approaches as compared to MLR model was assessed for modeling diagnostic measures. All methods provided unbiased estimates of diagnostic measures in the absence of any covariate. LR and GEE methods produced more biased estimates as compared to MLR approach especially for small sample size studies. No bias was obtained in predicting sensitivity measure using MLR method for one screening test. Our proposed MLR method is robust for modeling diagnostic measures of a screening test as opposed to LR method. MLR method and GEE method produced similar estimates of diagnostic measures for comparing two screening tests in large sample size studies. The proposed MLR model for diagnostic measures is simple, and available in common statistical software. Our study demonstrates that MLR method should be preferred as an alternative for modeling diagnostic measures.

Effective combined antiretroviral therapy (cART) in HIV-infected patients has made HIV a treatable infection; however, debilitating HIV-associated neurocognitive disorders (HAND) can still affect approximately 50% of HIV-infected individuals even under cART. While cART has greatly reduced the prevalence of the most severe form of HAND, milder forms have increased in prevalence, leaving the total proportion of HIV-infected individuals suffering from HAND relatively unchanged. In this study, an in vitro drug screen identified fluconazole and paroxetine as protective compounds against HIV gp120 and Tat neurotoxicity. Using an accelerated, consistent SIV/macaque model of HIV-associated CNS disease, we tested the in vivo neuroprotective capabilities of combination fluconazole/paroxetine (FluPar) treatment. FluPar treatment protected macaques from SIV-induced neurodegeneration, as measured by neurofilament light chain in the CSF, APP accumulation in axons, and CaMKIIα in the frontal cortex, but did not significantly reduce markers of neuroinflammation or plasma or CNS viral loads. Since HIV and SIV neurodegeneration is often attributed to accompanying neuroinflammation, this study provides proof of concept that neuroprotection can be achieved even in the face of ongoing neuroinflammation and viral replication.

The American College of Cardiology/American Society of Nuclear Cardiology published revised appropriate use criteria (AUC) for SPECT MPI in 2009. We assessed adherence to these guidelines and factors associated with inappropriate utilization at the University Medical Center. The AUC was applied retrospectively to 420 SPECT MPI studies. Two-sample t test, Fisher’s exact test, and multivariable logistic regression models were used for analysis. There were 322 appropriate (86%) and 54 (14%) inappropriate studies. The odds of having an inappropriate test increased with younger age (P

Following infection with Human immunodeficiency virus 1 (HIV-1) there is a remarkable variation in virus replication and disease progression. Both host and viral factors have been implicated in the observed differences in disease status. Here, we focus on understanding the contribution of HIV-1 viral protein R (Vpr) by evaluating the disease-associated Vpr polymorphism and its biological functions from HIV-1 positive rapid progressor (RP) and long-term nonprogressor (LTNP) subjects. Results presented here show distinct variation in phenotypes of Vpr alleles from LTNP and RP subjects. Most notably, the polymorphism of Vpr at R36W and L68M associated with RP shows higher levels of oligomerization, and increased virus replication, whereas R77Q exhibits poor replication kinetics. Interestingly, we did not observe correlation with cell cycle arrest function. Together these results indicate that polymorphisms in Vpr in part may contribute to altered virus replication kinetics leading to the observed differences in disease progression in LTNP and RP groups.

Studying the effect of exposure or intervention on a dichotomous outcome is very common in medical research. Logistic regression (LR) is often used to determine such association which provides odds ratio (OR). OR often overestimates the effect size for prevalent outcome data. In such situations, use of relative risk (RR) has been suggested. We propose modifications in Zhang and Yu and Diaz-Quijano methods. These methods were compared with stratified Mantel Haenszel method, LR, log binomial regression (LBR), Zhang and Yu method, Poisson/Cox regression, modified Poisson/Cox regression, marginal probability method, COPY method, inverse probability of treatment weighted LBR, and Diaz-Quijano method. Our proposed modified Diaz-Quijano (MDQ) method provides RR and its confidence interval similar to those estimated by modified Poisson/Cox and LBRs. The proposed modifications in Zhang and Yu method provides better estimate of RR and its standard error as compared to Zhang and Yu method in a variety of situations ...

Embryology is integrated into the Clinically Oriented Anatomy course at the Texas Tech University Health Sciences Center School of Medicine. Before 2008, the same instructor presented embryology in 13 face-to-face lectures distributed by organ systems throughout the course. For the 2008 and 2009 offerings of the course, a hybrid embryology instruction model with four face-to-face classes that supplemented online recorded lectures was used. One instructor delivered the lectures face-to-face in 2007 and by online videos in 2008-2009, while a second instructor provided the supplemental face-to-face classes in 2008-2009. The same embryology learning objectives and selected examination questions were used for each of the three years. This allowed direct comparison of learning outcomes, as measured by examination performance, for students receiving only face-to-face embryology instruction versus the hybrid approach. Comparison of the face-to-face lectures to the hybrid approach showed no difference in overall class performance on embryology questions that were used all three years. Moreover, there was no differential effect of the delivery method on the examination scores for bottom quartile students. Students completed an end-of-course survey to assess their opinions. They rated the two forms of delivery similarly on a six-point Likert scale and reported that face-to-face lectures have the advantage of allowing them to interact with the instructor, whereas online lectures could be paused, replayed, and viewed at any time. These experiences suggest the need for well-designed prospective studies to determine whether online lectures can be used to enhance the efficacy of embryology instruction.

To establish the relationship between HIV-induced cardiac diastolic dysfunction, immune responses, and virus replication in the heart using the simian immunodeficiency virus (SIV)/macaque model.Cardiac diastolic dysfunction is common in HIV-infected individuals including asymptomatic patients and those treated with combination antiretroviral therapy. SIV-infected macaques develop cardiac dysfunction, serving as a useful model to establish mechanisms underlying HIV-induced cardiac dysfunction. To understand the relationship between functional cardiac impairment, viral replication in the heart, and associated host inflammatory responses, cardiac function was evaluated in SIV-infected macaques and functional decline was correlated with features of the host immune response and the extent of viral replication in both the myocardium and plasma.Cardiac function was evaluated longitudinally in 22 SIV-infected and eight uninfected macaques using mitral inflow and tissue Doppler echocardiography. Myocardial macrophage populations were evaluated by CD68 and CD163 immunostaining. SIV RNA levels in both myocardium and plasma were measured by qRT-PCR.Echocardiographic abnormalities developed in SIV-infected macaques that closely resembled diastolic dysfunction reported in asymptomatic HIV-infected individuals. Although CD68 and CD163 were upregulated in the myocardium of SIV-infected animals, neither macrophage marker correlated with functional decline. SIV-induced diastolic dysfunction was strongly correlated with extent of SIV replication in the myocardium, implicating virus or viral proteins in the initiation and progression of cardiac dysfunction.This study demonstrated a strong correlation between cardiac functional impairment and extent of SIV replication in the myocardium, suggesting that persistent viral replication in myocardial macrophages induces cardiomyocyte damage manifest as diastolic dysfunction.

HIV-infected individuals, even with antiretroviral therapy, often display cognitive, behavioral and motor abnormalities and have decreased dopamine (DA) levels. Minocycline prevents encephalitis and neurodegeneration in SIV models, suggesting that it might also protect against nigrostriatal dopaminergic system dysfunction. Using an SIV/macaque model of HIV-associated CNS disease, we demonstrated that striatal levels of DA were significantly lower in macaques late in infection and that levels of the metabolite DOPAC also tended to be lower. DA levels declined more than its metabolites, indicating a dysregulation of DA production or catabolism. Minocycline treatment beginning at 12 but not 21 days postinoculation prevented striatal DA loss. DA decline was not due to direct loss of dopaminergic projections to the basal ganglia as there was no difference in tyrosine hydroxylase, dopamine transporter, vesicular monoamine transporter 2 or synaptophysin between minocycline-treated and untreated macaques. SIV-infected macaques had significantly higher monoamine oxidase (MAO) activity than uninfected macaques, although MAO activity was not affected by minocycline. Oxidative/nitrosative stress was examined by nitrotyrosine staining in the deep white matter and was lower in SIV-infected, minocycline-treated macaques compared with untreated macaques. These data suggest that minocycline, which has antioxidant activity, has a protective effect on DA homeostasis when administered at an appropriate time in SIV neuropathogenesis.

The lower gastrointestinal tract is a major mucosal site of HIV entry and initial infection. Thus, the induction of strong cellular immune responses at this mucosal site will be an important feature of an effective HIV vaccine. We have used a novel prime-boost vaccination approach to induce immune responses at mucosal sites. Orally delivered recombinant Clostridium perfringens expressing HIV-1 gag (Cp-Gag) was evaluated for induction of HIV-1 Gag specific T cell responses in a prime-boost model with intranasal inoculation of HIV-1 virus like particles (VLP). HIV-1 specific cellular immune responses in both the effector (Lamina propria) and inductive sites (Peyer's patches) of the gastrointestinal (GI) tract were significantly higher in mice immunized using Cp-Gag and VLPs in a prime-boost approach compared to mice immunized with either Cp-Gag or VLPs alone. Such cellular immune response was found to be mediated by both CD8(+) and CD4(+) T cells. Such a strong mucosal immune response could be very useful in developing a mucosal vaccine against HIV-1.

Peripheral neuropathy is the most common neurological complication of HIV-1 infection, affecting over one-third of infected individuals, including those treated with antiretroviral therapy. To study the pathogenesis of HIV-induced peripheral nervous system disease, we established a model in which SIV-infected macaques developed changes closely resembling alterations reported in components of the sensory pathway in HIV-infected individuals. Significant declines in epidermal nerve fiber density developed in SIV-infected macaques, similar to that of HIV-infected individuals with neuropathy. Changes in dorsal root ganglia (DRG) included macrophage infiltration, SIV replication in macrophages, immune activation of satellite cells, and neuronal loss. To determine whether dorsal root ganglion damage was associated with altered nerve function, we measured unmyelinated C-fiber conduction velocities (CV) in nerves of SIV-infected macaques and compared CV changes with DRG alterations. Twelve weeks postinoculation, SIV-infected macaques had significantly lower C-fiber conduction velocity in sural nerves than uninfected animals and the magnitude of conduction velocity decline correlated strongly with extent of DRG macrophage infiltration. Thus, injury to neurons in the DRG—mediated by activated macrophages—preceded altered conduction of unmyelinated nerve fibers in SIV-infected macaques, suggesting that macrophage-mediated DRG damage may be the initiating event in HIV-induced sensory neuropathy.

Infection by HIV-1 frequently leads to pulmonary complications, including alterations to local immune environments. To better understand these alterations, we have examined in detail the patterns and levels of expression of chemokine, cytokine, and chemokine receptor mRNAs in lung tissues from 16 uninfected or simian immunodeficiency virus (SIV)/DeltaB670 infected cynomolgus macaques at different stages of infection. Among the most up-regulated immune genes were interferon (IFN)-γ, IFN-γ-inducible CXCR3 ligands, and CCR5 ligands, as well as the cognate chemokine receptors. These changes were greatest in animals with clear Pneumocystis carinii coinfection. Immunohistochemistry and in situ hybridization revealed monocytes/macrophages to be the predominant type of cell infiltrating into lung tissues and serving as the major cellular source of chemokines. To explore the causes of chemokine alterations, we treated macaque lung cells with IFN-γ, lipopolysaccharide, Poly(I:C), and P. carinii in vitro, and results revealed that these stimuli can induce the expression of CXCR3 ligand and/or CCR5 ligand mRNAs. Taken together, these studies provide a comprehensive definition of the chemokine networks available to modulate cellular recruitment to lung tissues during SIV infection and implicate both cytokines (IFN-γ) and pathogens (SIV and P. carinii) as contributors to increased expression of pro-inflammatory chemokines.

Background During the era of highly active antiretroviral therapy (HAART), the prevalence of HIV-associated central nervous system (CNS) disease has increased despite suppression of plasma viremia. Methods In a simian immunodeficiency virus (SIV) model system in which all animals develop AIDS and 90% develop CNS disease by 3 months after inoculation, pigtailed macaques were treated with a regimen of tenofovir disoproxil fumarate, saquinavir, atazanavir, and an integrase inhibitor starting at 12 days after inoculation and were euthanized at approximately 175 days after inoculation. Results Plasma and cerebrospinal fluid (CSF) viral loads declined rapidly after the initiation of HAART. Brain viral RNA was undetectable at necropsy, but viral DNA levels were not different from those in untreated SIV-infected macaques. CNS inflammation was significantly reduced, with decreased brain expression of major histocompatibility complex class II and glial fibrillary acidic protein and reduced levels of CSF CCL2 and interleukin 6. Brain from treated macaques had significantly lower levels of interferon beta, type 1 interferon-inducible gene myxovirus (influenza) resistance A, and indolamine 2,3-dioxygenase messenger RNA, suggesting that immune hyperactivation was suppressed, and fewer CD4(+) and CD8(+) T cells, suggesting that trafficking of T cells from peripheral blood was reduced. Brain levels of CD68 protein and tumor necrosis factor alpha and interferon gamma RNA were reduced but were not significantly lower, indicating continued CNS inflammation. Conclusions These data, generated in a rigorous, high-viral-load SIV-infected macaque model, showed that HAART provided benefits with respect to CNS viral replication and inflammation but that no change in the level of viral DNA and continued CNS inflammation occurred in some macaques.

Biologic and genetic differences between HIV-1 clade C in India and clade B in US suggest that the effect of anti-viral therapy in various body compartments may differ between these two clades. We examined the effect of therapy on viral loads in semen and blood of HIV-1-clade C infected subjects from India and evaluated whether HIV-1 in the semen is different from that in blood in these subjects. HIV-1 RNA was detected in semen and blood at all stages of the disease. Viral loads in semen and blood were strongly correlated with each other, but not with the CD4+ T cell count. Anti-viral treatment reduced viral load drastically in blood and semen within one month of post therapy. Genetic characterization of HIV-1 in the semen and blood demonstrated that they were highly compartmentalized. These data have important implications of sexual transmission of HIV-1 in clade C HIV-1 infected subjects.

The molecular mechanisms underlying learning and memory impairment in patients with HIV-associated neurological disease have remained unclear. Calcium/calmodulin-dependent kinase II (CaMKII) has key roles in synaptic potentiation and memory storage in neurons and also may have immunomodulatory functions. To determine whether HIV and simian immunodeficiency virus (SIV) induce alterations in CaMKII expression and/or activation (autophosphorylation) in the brain, we measured CaMKII alterations by quantitative immunoblotting in both an in vitro HIV/neuronal culture model and in vivo in an SIV-infected macaque model of HIV-associated neurological damage. Using primary rat hippocampal neuronal cultures treated with culture supernatants harvested from HIV-1–infected human monocyte-derived macrophages (HIV/MDM), we found that CaMKII activation declined after exposure of neurons to HIV/MDM. Consistent with our in vitro measurements, a significant decrease in CaMKII activation was present in both the hippocampus and frontal cortex of SIV-infected macaques compared with uninfected animals. In SIV-infected animals, total CaMKII expression in the hippocampus correlated well with levels of synaptophysin. Furthermore, CaMKII expression in both the hippocampus and frontal cortex was inversely correlated with viral load in the brain. These findings suggest that alterations in CaMKII may compromise synaptic function in the early phases of chronic neurodegenerative processes induced by HIV.

Staphylococcus aureus (SA) nasal carriage is transient in most humans and usually benign, but dissemination of SA to extranasal sites causes the majority of clinical infections, and SA is a major cause of serious infections in the U.S. A better understanding of innate nasal decolonization mechanisms is urgently needed, as are relevant models for studying SA clearance. Here, we screened a population of healthy smokers for nasal SA carriage, and compared participants’ abilities to clear experimentally applied nasal SA before and after completion of a smoking cessation program. We determined that cigarette smoking increases mean nasal SA load (2.6×104 CFU/swab) compared to healthy non-smokers (1.7×103 CFU/swab) and might increase the rate of SA nasal carriage in otherwise healthy adults: 22 of 99 smokers carried SA at the screening visit, while only 4 of 30 non-smokers screened positive during the same time period. Only 6 of 19 experimental inoculation studies in active smokers resulted in SA clearance within the month of follow-up, while in the cessation group, 6 of 9 subjects cleared nasal SA and carriage duration averaged 21±4 days. Smoking cessation associated with enhanced expression of SA-associated IL-1β and G-CSF in nasal fluids. Participants who failed to clear SA exhibited higher nasal SA load and elevated nasal interleukin-1 receptor antagonist (IL-1RA) expression at the pre-experiment study visits. We conclude that smokers exhibit higher SA load than non-smokers, and that innate immune pathways including G-CSF expression and signaling through the IL-1 axis are important mediators of nasal SA clearance.

Inhalation of airborne microorganisms and organic dust is an occupational concern among workers in agricultural industries. Airborne microorganisms and particulate matter samples were collected from poultry house, flourmill, textile, and food industry sites by use of liquid impinger and gravimetric samplers. Particulate matter concentrations were recorded at median concentrations of 1.56, 1.92, 4.39, and 0.7 mg/m3 in the occupied poultry house, textile, flourmill, and food indoor working environments, respectively. The highest median particulate matter concentration (27.9 mg/m3) was detected at the flourmill’s stack site. The highest median indoor concentration of culturable airborne bacteria (6.23 × 105 CFU/m3) was found at the occupied poultry-house site and the lowest concentration (4.6 × 103 CFU/m3) was found at the food industry site. The highest median indoor concentration of culturable airborne fungi (3.15 × 104 CFU/m3) was found at the flourmill site whereas the lowest (1.24 × 103 CFU/m3) was found at the textile industry site. Bacillus and Staphylococcus were the predominant Gram-positive bacteria whereas Acinetobacter and Klebsiella were the predominant Gram-negative bacteria. Escherichia coli and Salmonella were only detected in the indoor air at the poultry house site. Aspergillus flavus, Aspergillus niger, Penicillium, and yeast were the predominant fungal types at flourmill, textile, food industry, and poultry house, respectively. Workers were continuously exposed to airborne microorganisms at a median value of 104 CFU/m3 in all the industries studied.

To characterize the regenerative pattern of cutaneous nerves in simian immunodeficiency virus (SIV)-infected and uninfected macaques, excisional axotomies were performed in nonglabrous skin at 14-day intervals. Samples were examined after immunostaining for the pan-axonal marker PGP 9.5 and the Schwann cell marker p75 nerve growth factor receptor. Collateral sprouting of axons from adjacent uninjured superficial dermal nerve bundles was the initial response to axotomy. Both horizontal collateral sprouts and dense vertical regeneration of axons from the deeper dermis led to complete, rapid reinnervation of the epidermis at the axotomy site. In contrast to the slower, incomplete reinnervation previously noted in humans after this technique, in both SIV-infected and uninfected macaques epidermal reinnervation was rapid and completed by 56 days postaxotomy. p75 was densely expressed on the Schwann cells of uninjured nerve bundles along the excision line and on epidermal Schwann cell processes. In both SIV-infected and uninfected macaques, Schwann cell process density was highest at the earliest timepoints postaxotomy and then declined at a similar rate. However, SIV-infection delayed epidermal nerve fiber regeneration and remodeling of new sprouts at every timepoint postaxotomy, and SIV-infected animals consistently had lower mean epidermal Schwann cell densities, suggesting that Schwann cell guidance and support of epidermal nerve fiber regeneration may account for altered nerve regeneration. The relatively rapid regeneration time and the completeness of epidermal reinnervation in this macaque model provides a useful platform for assessing the efficacy of neurotrophic or regenerative drugs for sensory neuropathies including those caused by HIV, diabetes mellitus, medications, and toxins.

Objective. Sleep disturbance is common in the subacute recovery phase following brain injury. A previous study from the authors' group found 68% of patients with closed head injury (CHI) had disrupted sleep on a rehabilitation unit. In the present study, the authors investigated whether improvement in sleep efficiency correlates with duration of posttraumatic amnesia (PTA) after CHI. Methods. Fourteen CHI patients were enrolled and followed prospectively. Mechanism of injury included motor vehicle accident, fall, and blunt assault. An actigraph was placed on each subject's wrist within 72 hours of admission to the rehabilitation unit and recorded data for the duration of their stay. A minimum of 7 days of continuous actigraphy data was obtained on all subjects. PTA was measured daily using the Orientation Log (O-LOG). Results. Seventy-eight percent of subjects had mean week-1 sleep efficiency scores of ≤63%. Patients admitted having already cleared PTA had significantly better week-1 sleep efficiency scores than those with ongoing amnesia ( P = .032). For those patients admitted with ongoing PTA, each 10-unit increase in sleep efficiency score correlated with 1 unit increase in O-LOG score ( P = .056). Conclusions. Disrupted sleep is common in the postacute stage following CHI. Improved sleep efficiency correlates with resolution of PTA. Decreased sleep efficiency may negatively affect memory return after traumatic brain injury. Actigraphy is uniquely suited to study the sleep patterns of these patients.

This study was undertaken to determine the normal indoor and outdoor airborne culturable fungal constituents and concentrations of an arid environment. Air samples were taken with two-stage, ambient, culturable sampler systems and analyzed for nine specific fungal genera from 50 homes as a repeated measure during each season of the year. These homes had no previous histories of indoor air quality issues. This study detected seasonal differences for the arid environment between different culturable fungal concentrations across the two size ranges. The highest concentrations were during fall, in the outdoor fine-size range. The lowest concentrations were the indoor coarse concentrations in the spring. From this study it can be concluded that Cladosporium spp. had the highest concentrations during fall in an arid environment. The overall findings suggest that Cladosporium had concentrations greater than the other genera evaluated, specifically, the fall outdoor fine concentrations. Seasonality was found to be a key factor in determining the variability of fungal constituents and concentrations within the arid indoor and outdoor environments. The fine-size range was 12 times and 6 times greater than the coarse-size range for indoor and outdoor samples, respectively, which accounted for the majority of fungal organisms. In addition, the results from this study in an arid climate differ from those conducted in a moister climate.

Regulatory T cells (Treg) play key roles in immune regulation through multiple modes of suppression. The effects of HIV-1 infection on Treg levels in lymphoid tissues remain incompletely understood. To explore this issue, we have measured the levels of forkhead box protein 3 (FOXP3)-positive cells and associated immunomodulatory genes in a pathogenic simian immunodeficiency virus (SIV)/macaque model and found that a loss of Treg in lymph nodes (LNs) occurred following SIV infection. Changes in expression of the ligands for CXCR3, CCR4, and CCR7, and the cytokines TGF-β and IL-2 were all linked to this loss of Treg, which in turn was linked with increased levels of cellular activation. Our findings identify three mechanisms that likely contribute to SIV-driven loss of Treg, including reduced levels of cytokines associated with Treg differentiation and altered expression of agonist and antagonist chemokines. The loss of Treg and the associated cellular activation in lymphoid tissues is consistent with the events in HIV-1 infected individuals and suggest that components of the Treg differentiation and trafficking network could be targets for therapeutic intervention. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.

Traumatic brain injury (TBI) is a leading cause of disability in young people in the United States. Disorders of arousal and attention are common in closed head injury (CHI). Daytime drowsiness impairs participation in rehabilitation, whereas nighttime wakefulness leads to falls and behavioral disturbances. Sleep disturbances in TBI reported in the literature have included excessive daytime somnolence, sleep phase cycle disturbance, narcolepsy, and sleep apnea. Although well known to the clinician treating these patients, the extent and prevalence of disrupted sleep in patients in an acute inpatient rehabilitation unit has not been described. Objective. To determine the prevalence of sleep wake cycle disturbance (SWCD) in patients with CHI in a TBI rehabilitation unit. Design. Prospective observational. Setting. Inpatient specialized brain injury rehabilitation unit. Patients. Thirty-one consecutive admissions to a brain injury rehabilitation unit with the diagnosis of CHI. Results. Twenty-one patients (68%) had aberrations of nighttime sleep. There was no significant difference in Glasgow Coma Score on admission to trauma nor was there any significant difference in age between the affected and unaffected groups. Patients with SWCD had longer stays in both the trauma center ( P < .003) and the rehabilitation center ( P < .03). Conclusions. There is a high prevalence of SWCD in CHI patients admitted to a brain injury rehabilitation unit. Patients with SWCD have longer stays in both acute and rehabilitation settings and may be a marker for more severe injury.

Peripheral neuropathy is the most frequent neurologic complication associated with human immunodeficiency virus (HIV) infection, yet its pathogenesis remains poorly understood. To study the mechanisms causing HIV-induced peripheral nervous system disease, we examined trigeminal ganglia obtained from simian immunodeficiency virus (SIV)-inoculated macaques. SIV-infected macaques developed multifocal trigeminal ganglionitis of varying severity characterized by multifocal mononuclear infiltrates, neuronophagia, and neuronal loss resembling reports of HIV-associated changes present in dorsal root ganglia. Neuronal density, measured by calculating the fractional area of trigeminal ganglia occupied by neurons, was significantly lower in SIV-infected macaques versus uninfected macaques (p = 0.001). To characterize the inflammatory cell population and measure productive viral infection in ganglia, trigeminal ganglia from SIV-infected macaques were immunostained for macrophage or cytotoxic lymphocyte markers and for SIV gp41. The extent of macrophage infiltration in trigeminal ganglia was inversely correlated with neuronal loss (p = 0.001), whereas cytotoxic lymphocyte infiltration was not associated with neuronal loss. These studies demonstrate that alterations in the somatosensory ganglia of SIV-infected macaques closely parallel those observed in HIV-infected individuals and show that study of SIV-infected macaques may help elucidate the pathophysiology of HIV-induced peripheral neuropathy.

The “shock and kill” model of human immunodeficiency virus type 1 (HIV-1) eradication involves the induction of transcription of HIV-1 genes in latently infected CD4+T cells, followed by the elimination of these infected CD4+T cells by CD8+T cells or other effector cells. CD8+T cells may also be needed to control the spread of new infection if residual infected cells are present at the time combination antiretroviral therapy (cART) is discontinued. In order to determine the time frame needed for CD8+T cells to effectively prevent the spread of HIV-1 infection, we examined the kinetics of HIV transcription and virus release in latently infected cells reactivatedex vivo. Isolated resting, primary CD4+T cells from HIV-positive (HIV+) subjects on suppressive regimens were found to upregulate cell-associated HIV-1 mRNA within 1 h of stimulation and produce extracellular virus as early as 6 h poststimulation. In spite of the rapid kinetics of virus production, we show that CD8+T cells from 2 out of 4 viremic controllers were capable of effectively eliminating reactivated autologous CD4+cells that upregulate cell-associated HIV-1 mRNA. The results have implications for devising strategies to prevent rebound viremia due to reactivation of rare latently infected cells that persist after potentially curative therapy.IMPORTANCEA prominent HIV-1 cure strategy termed “shock and kill” involves the induction of HIV-1 transcription in latently infected CD4+T cells with the goal of elimination of these cells by either the cytotoxic T lymphocyte response or other immune cell subsets. However, the cytotoxic T cell response may also be required after curative treatment if residual latently infected cells remain. The kinetics of HIV-1 reactivation indicate rapid upregulation of cell-associated HIV-1 mRNA and a 5-h window between transcription and virus release. Thus, HIV-specific CD8+T cell responses likely have a very short time frame to eliminate residual latently infected CD4+T cells that become reactivated after discontinuation of antiretroviral therapy following potentially curative treatment strategies.

Background Before the onset of AIDS, replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) in the lungs is considered to be latent. When and how virus replication is controlled in the lungs is unclear. In the present study, we examine virus replication in the lungs and in cells recovered from bronchoalveolar lavage (BAL) samples in a comprehensive, longitudinal analysis of an SIV/macaque model. Methods Gene-specific RNA and DNA were quantitated by polymerase chain reaction (PCR) and by real-time reverse-transcription PCR (RT-PCR). Alveolar macrophages were isolated using Dynabeads CD14 (Invitrogen). Expression of CCAAT/enhancer-binding protein beta (C/EBP beta ) isoforms was examined by Western blot analysis. Results SIV replication occurred in the lungs during acute infection and correlated with plasma viral load. Innate immune responses involving interferon- beta and the dominant-negative isoform of C/EBP beta were induced at this time. SIV RNA expression was suppressed in the lungs during asymptomatic infection, when no correlation existed with plasma viral load until SIV RNA levels rebounded again during late-stage disease. Modulation of viral RNA levels in BAL cells reflected RNA levels in lung tissue throughout each phase of infection. Conclusion Quantitation of SIV RNA in BAL cells provides a consistent surrogate assessment of virus replication in lung tissue. Innate immune responses contribute to compartmentalized suppression of acute SIV replication in the lungs.

Pap smear testing continues to be the single most effective tool in reducing deaths due to cervical cancer (Watkins, Gabali, Winkleby, Gaona & Lebaron, 2002). Despite the creation of a national cervical cancer screening program, more than 4,000 women die every year in Mexico from this disease. This study explored the knowledge, attitudes, and behaviors of Mexican women regarding cervical cancer screening, and identified beliefs and barriers that may influence cervical health. All analyses compared women who had ever had a Pap test in their lives (“ever”) with women who had never had a Pap test (“never”). In bivariate analyses, the following variables were significantly associated (p

Objective: To compare the viral genotypes present in RNA from brain and peripheral blood mononuclear cells (PBMC) and DNA from brain during acute, asymptomatic and late stages of SIV infection of macaques. Methods: Eighteen pigtailed macaques were intravenously inoculated with SIV. At 10, 21 and 56 days postinoculation, six were euthanized and the severity of encephalitis was assessed by microscopic examination. DNA and RNA were isolated from brain and PBMC, and the V1 region of env was amplified by the polymerase chain reaction and sequenced from over 800 different clones. Results: Similar genotypes were detected in RNA from brain and PBMC at 10 days postinoculation, suggesting an unrestricted exchange of virus between the periphery and the brain during acute infection. There was a progressive increase in the percentage of neurovirulent genotypes in brain RNA from acute (14% of all genotypes detected in brain RNA) to early asymptomatic (45%), to late asymptomatic (52%) and to terminal (95%) infection. Fewer different genotypes were found in brain RNA than in PBMC RNA from macaques euthanized during early asymptomatic (2.5 and 5 different genotypes, respectively; P = 0.007), late asymptomatic (2 and 5 different genotypes, respectively; P = 0.003) and terminal (2 and 4 different genotypes, respectively; P < 0.001) infection. Conclusion: These data demonstrate that the almost exclusive replication of neurovirulent genotypes in the brain seen at late-stage infection is a progressive process that begins early in infection and continues to late stage disease.

HIV testing and counseling has been associated with reductions in risk behaviors in some populations. This study examined whether involvement in a long-term study, including exposure to repeated HIV testing and counseling, was associated with increased condom use among injection drug users (IDUs) through a retrospective analysis of an IDU cohort from Baltimore, MD, during 1994-1998. Eligibility included being aged 18 years or older, injecting within 10 years and not having initiated antiretroviral therapy. A logistic model of high versus low risk, based on condom use, was used. Of 322 eligible IDUs, most were male (66%) and African-American (94%). No significant change in the odds of inconsistent condom use was observed with continued study exposure. Condom use remained low, indicating a need for interventions to reduce sexual risks and HIV transmission in this population.

Objectives: The objective of this research was to explore prenatal smoking behaviors among Black women attending prenatal clinics. Despite the racial disparities in poor pregnancy outcomes, and the well-known association of smoking with harmful outcomes, little research has been conducted about prenatal smoking among Black women. Methods: Women were enrolled in the study and interviewed at the time of the first prenatal visit. The interview contained items to assess prenatal smoking and cessation, depressive symptoms, demographic factors, and beliefs about smoking. Reports of smoking cessation were verified using urinary cotinine. Results: The sample consisted of 811 Black women. Fourteen percent of the women were self-reported smokers, 12.6% reported cessation and 73% were nonsmokers. Twenty percent of the self-reported quitters had elevated cotinine; when these women were reclassified, 17% of the women were smokers. Factors associated with smoking in logistic regression analysis included elevated maternal depressive symptoms (OR = 1.7, 95% CI: 1.1–2.6), maternal age 20 years or older (OR = 1.94; 95% CI: 1.1, 3.3), less than a high school education (OR = 2.2; 95% CI: 1.2, 3.8), unmarried/not living with a partner (OR = 1.9; 95% CI: 1.0, 3.6), and allowing smoking in the home (OR = 5.5; 95% CI: 3.4, 8.6). Conclusions: The prevalence of maternal prenatal smoking was much higher among women in this sample than has been previously reported. The rate of nondisclosure of smoking among self-reported quitters was also high. Maternal behavioral (allowing smoking in the home) and psychosocial factors (depressive symptoms) were associated with prenatal smoking.

We characterized factors and temporal trends associated with discontinuation of highly active antiretroviral therapy (HAART) among 936 HIV-infected women enrolled in the Women's Interagency HIV Study. A multivariate analysis of post-HAART initiation exposures found that high HIV RNA levels (relative hazard [RH] = 1.36, P < 0.001) and high depressive symptom scores (RH = 1.53, P = 0.012) were associated with HAART discontinuation. The adjusted hazard of discontinuation was higher in the 2 most recent calendar periods compared with the first (RH = 1.61, P = 0.026; RH = 1.56, P = 0.074, respectively). The increasing risk of HAART discontinuation in recent calendar periods and changes in the clinical factors associated with discontinuation reflect ongoing and dynamic shifts in the approach to HAART utilization.

Three-dimensional (3-D) bar graphs and their current 2-D alternatives have certain limitations when the primary objective is to provide an equal representation of the effects of two predictors on an outcome. This article proposes a graphing methodology (in the shape of a diamond) that projects 3-D bar graphs into 2-D whereby the third dimension is replaced with a polygon whose area and middle vertical and horizontal lengths represent the outcome. The proposed graphical representation is invariant to rotations and avoids outcomes in categories being concealed by others. This article shows several applications of our proposal to a variety of data types (e.g., proportions, incidence rates, relative risks), which can be easily implemented with available software.

Human immunodeficiency virus type 1 (HIV-1) currently infects ∼40 million people worldwide [1]. More than 95% of all new cases occur in developing countries, with heterosexual transmission being the predominant route of infection [1]. Rates of HIV-1 infection continue to increase, particularly among nonwhites and women [1]. In sub-Saharan Africa, a region that accounts for 70% of infected individuals globally, women with HIV-1 now outnumber men with HIV-1 [1]. Host differences may contribute to the rapid spread of HIV-1 in some areas of the world; specific genetic traits have been found to influence susceptibility to HIV-1 transmission and disease progression. In addition to CD4, the primary HIV-1 receptor, a functional secondary receptor is required to initiate infection. Human β-chemokine receptors serve this function, with CCR5 serving as coreceptor for macrophage-tropic strains of HIV-1 [2]. A 32-bp deletion (Δ32) in the CCR5 gene renders this receptor nonfunctional, and cells that are homozygous for this mutation are highly resistant to infection by macrophage-tropic strains of HIV-1 [3]. The mutation is most common in white populations, where the frequency of the Δ32 allele ranges from 4% to 16%; ∼20% of whites are heterozygous, and 1% are homozygous for Δ32 [4]. However, this allele is less common among US blacks and Latinas and was absent in Africans and Asian populations studied in anthropological surveys and investigations of non–HIV-related conditions [4]. Investigation of large cohorts of individuals exposed sexually or parenterally to HIV-1 found that the homozygous Δ32 mutation confers a high level of resistance to infection [5–8]. These studies, primarily composed of white men, also found that Δ32 heterozygotes exhibit delayed HIV-1 disease progression but were not protected against transmission [6–8]. However, a more recent study of high-risk HIV-1–seronegative men did demonstrate partial protection against HIV-1 infection in those with the Δ32 heterozygous genotype [9]. Because Δ32 heterozygotes of either sex express a reduced number of CCR5 receptors, compared with individuals with the wild-type genotype, the partial protection and delayed disease progression in heterozygotes may be related to the decreased number of CCR5 receptors and the resulting decreased virus load. Transmission studies involving women showed variable results; 2 studies suggested that the heterozygous state confers partial protection against HIV-1 infection [5, 10], whereas 3 other studies did not [9, 11, 12]. However, all the previous analyses had a suboptimal statistical power for assessing this question, with no more than 630 women in each study. Recently, investigators constructed a mathematical model of the dynamics of HIV-1 transmission in populations with or without the Δ32 mutation. This model predicted that the Δ32 allele would slow the heterosexual spread of the epidemic in populations where the mutation was common [13]. This prediction, coupled with the results from previous cohort studies, points out the importance of studying the role of the Δ32 deletion in HIV-1 transmission in a larger cohort of women. Studies have documented sex-specific differences in HIV-1 transmission, infection, and disease progression [10, 14–18]. Because the routes and mechanisms of HIV-1 infection may differ in significant ways between men and women, we investigated the relationship of CCR5 genotype to the likelihood of HIV-1 infection among women enrolled in the Women's Interagency HIV Study (WIHS), a large study of HIV-1 infection in women conducted in 5 US cities.

Objectives As the U.S. economy began its downward trend in 2008, many citizens lost their jobs and, ultimately, their employer-sponsored health care insurance. The expectation was that many of the newly uninsured would turn to emergency departments (EDs) for their health care. This study was undertaken to determine, first, if changes in the insurance status of the general population were reflected in the ED insurance payer mix and, second, whether there was evidence of an increased reliance on the ED as a continuing source of health care for any payer group(s). Methods This was a retrospective observational study using public data files from the National Hospital Ambulatory Medical Care Survey for Emergency Departments for years 2006 through 2010 (2008 ± 2 years). Changes in the relative proportions of ED visits funded annually by private insurance, Medicaid, Medicare, and self-pay (uninsured) were analyzed using a logistic model. Poisson regression was used to compare trends in the rates of ED visits for each payer type (i.e., number of ED visits per 100 persons with each insurance type). A linear spline term was used to determine if there was a change in each risk estimate after 2008 compared to the risk estimate before 2008. Results Before 2008, the odds of an ED visit being funded by private insurance increased by 4% per year (odds ratio [OR] = 1.04, 95% confidence interval [CI] = 0.98 to 1.10; p = 0.15), but after 2008 the odds reversed, decreasing by nearly 10% per year (OR = 0.91, 95% CI = 0.85 to 0.97; p = 0.02). Medicaid-funded visits demonstrated opposite trends with a small decreasing trend of 2% per year before 2008 (OR = 0.98, 95% CI = 0.92 to 1.04; p = 0.52), followed by a significantly increasing trend of 20% per year after 2008 (OR = 1.20, 95% CI = 1.12 to 1.27; p = 0.001). The growth in Medicaid-funded ED visits was attributable to increased numbers of visits by both pediatric (

Background Diastolic dysfunction is a highly prevalent cardiac abnormality in asymptomatic as well as ART ‐treated human immunodeficiency virus ( HIV ) patients. Although the mechanisms underlying depressed cardiac function remain obscure, diastolic dysfunction in SIV ‐infected rhesus macaques is highly correlated with myocardial viral load. As cardiomyocytes are not productively infected, damage may be an indirect process attributable to a combination of pro‐inflammatory mediators and viral proteins. Methods and Results Given the diverse roles of CCR5 in mediating recruitment of leukocytes to inflammatory sites and serving as a receptor for HIV entry into cells, we investigated the role of CCR5 in the SIV/macaque model of diastolic dysfunction. We found that in SIV‐infected macaques, CCR5 inhibition dramatically impacted myocardial viral load measured by qRT ‐PCR and prevented diastolic dysfunction measured by echocardiography. Complementary in vitro experiments using fluorescence microscopy showed that CCR5 ligands impaired contractile function of isolated cardiomyocytes, thus identifying CCR5 signaling as a novel mediator of impaired cardiac mechanical function. Conclusions Together, these findings incriminate SIV / HIV gp120‐ CCR 5 as well as chemokine‐ CCR 5 interactions in HIV ‐associated cardiac dysfunction. These findings also have important implications for the treatment of HIV ‐infected individuals: in addition to antiviral properties and reduced chemokine‐mediated recruitment and activation of inflammatory cells, CCR 5 inhibition may provide a cardioprotective benefit by preventing cardiomyocyte CCR 5 signaling.

Macrophage chemoattractant protein–1 (MCP-1) may be a key trigger for the influx of macrophages into the brain in human immunodeficiency virus (HIV) encephalitis. In this study, simian immunodeficiency virus–infected macaques that developed moderate-to-severe encephalitis had significantly higher MCP-1 levels in cerebrospinal fluid (CSF) than in plasma as early as 28 days after inoculation, which was before the development of brain lesions. In contrast, CSF:plasma MCP-1 ratios remained constant at preinoculation levels in macaques that developed minimal or no encephalitis. Abundant MCP-1 protein and mRNA were detected in both macrophages and astrocytes in the brain. Macaques with increased MCP-1 in CSF had significantly greater expression of markers of macrophage and microglia activation and infiltration (CD68; ) and astrocyte activation (glial fibrillary acidic protein; P p .003 and in white and gray matter, respectively). The results suggest that the P p .019 P p .031 CSF:plasma MCP-1 ratio may be a valuable prognostic marker for the development of HIVinduced central nervous system disease. About 25% of untreated human immunodeficiency virus (HIV)‐infected individuals and 15% of individuals treated with highly active antiretroviral therapy develop dementia, whereas a larger percentage develop HIV encephalitis [1‐4]. High virus load in the cerebrospinal fluid (CSF) identifies people with active HIV replication in the brain, but many of those individuals already have ongoing inflammatory changes in the brain or HIV-associated cognitive or motor deficits (or both) [5‐9]. Early identification of individuals with HIV encephalitis would permit the earlier use of antiretroviral drugs that cross the bloodbrain barrier. These drugs may be more effective in suppressing central nervous system (CNS) inflammation, thus reducing the risk of HIV-associated dementia. HIV encephalitis is characterized by activation of glial cells, including astrocytes and microglia, and infiltration of inflammatory cells, particularly macrophages, into the brain [10, 11].

We evaluated CD4 cell counts over a 3(1/2) year period following the initiation of potent antiretroviral therapy (ART) in the Multicenter AIDS Cohort Study. The study population included 314 HIV-infected gay men who provided CD4 cell counts for at least 2 years after the initiation of potent ART. Trends in CD4 cell counts and plasma HIV-RNA were analyzed by regression methods that incorporated the statistical dependencies of outcomes measured over time within individuals. Regardless of CD4 cell count at initiation of potent ART, CD4 cell counts increased significantly (p